Recent studies have shown an overexpression of nucleoside transporters in some human breast, liver, stomach and colorectal cancer tissues, as well as varying profiles of nucleoside transporter subtype gene overexpression among tumors. This might underscore the marked increases in the capacity to take up physiological nucleosides that has been observed as normal cells are transformed into tumor cells. It has also been revealed that nucleoside transporter genes belong to the delayed early proliferative genes class. Taken together, these observations strongly point to a possible role of nucleoside transporters in the carcinogenesis process, at least at the tumor promotion and progression stages. We have recently found that nucleoside transport inhibitors potently antagonize the inhibitory effect of exogenous nucleosides on the antitumor promotion activity of dehydroepiandrosterone (DHEA) against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in the JB6 cell carcinogenesis model. This suggests that nucleoside transporters may be involved in the establishment of the tumorigenic phenotype, i.e. promotion of anchorage independent growth characteristics. This is consistent with reports that chemopreventive agents like tamoxifen downregulate nucleoside transporter gene expression. The long-term goal of this research program is to investigate the chemopreventive potential of nucleoside transport inhibitors. The specific aims of this project are: 1) to probe the changes in nucleoside transporter gene expression during JB6 cell tumorigenic transformation, 2) to determine whether nucleoside transport blockers can inhibit carcinogenesis compete against the inhibitory effects of exogenous nucleoside supplies on chemopreventive activity, and 3) to determine structure-activity relationships (SAR) regarding chemoprevention and inhibition of nucleoside transport. The research plan involves experiments to define the role of nucleoside transport inhibition in modulating the effects of chemopreventive agents like DHEA and tamoxifen, in the in vitro JB6 cell carcinogenesis model. Nucleoside transport inhibitors, namely, nitrobenzylthioinosine, dipyridamole and dilazep, as well as novel tetrahydroisoquinolinyl purine riboside inhibitors to be synthesized in the PI's laboratory will be used in these studies. The TPA-induced JB6 P+ cell transformation model using anchorage independent growth as end point, as well as JB6 P+/AP-l-luciferase reporter cell tumor promoter models will be employed for these proof-of-concept in vitro studies. Quantitative analysis of nucleoside transporter gene expression during JB6 tumorigenic transformation will also be carried out by means of real time quantitative RT-PCR. Ancillary studies involving analysis of cellular nucleotide levels by HPLC, and assays of G6PDH activity and its inhibition by DHEA will also be conducted. The mechanistic insights to be gained regarding the carcinogenesis process and its abrogation may lead to the identification of novel molecular targets and agents for cancer prevention.